Phylogeography, pre-zygotic isolation and taxonomic status in the

Transcription

Phylogeography, pre-zygotic isolation and taxonomic status in the
J Ornithol (2012) 153:303–312
DOI 10.1007/s10336-011-0744-8
ORIGINAL ARTICLE
Phylogeography, pre-zygotic isolation and taxonomic status
in the endemic Cyprus Wheatear Oenanthe cypriaca
Christoph Randler • Marc I. Förschler
Javier Gonzalez • Mansour Aliabadian
Franz Bairlein • Michael Wink
•
•
Received: 13 April 2010 / Revised: 1 April 2011 / Accepted: 10 August 2011 / Published online: 6 September 2011
Ó Dt. Ornithologen-Gesellschaft e.V. 2011
Abstract The insular endemic Cyprus Wheatear Oenanthe cypriaca has been considered as a subspecies of Pied
Wheatear O. pleschanka. However, due to several differences in behaviour, habitat selection and morphology, it is
currently treated by most authors as an independent species. Here, we used mitochondrial nucleotide sequences of
the cytochrome oxidase subunit 1 gene (679 base pairs),
playback experiments and dummy presentations to assess
the status of O. cypriaca. For the playback experiments we
used the conspecific song, and heterospecific songs of the
two subspecies of Black-eared Wheatear O. hispanica
hispanica and O. hispanica melanoleuca, O. pleschanka,
and Finsch’s Wheatear O. finschii. Experimental dummy
presentations included O. cypriaca, O. pleschanka and a
dark and light morph of O. h. melanoleuca. O. cypriaca
responded significantly stronger towards the conspecific
model and towards conspecific playbacks than towards
heterospecific stimuli. ML and BI analyses support the
close relationship between O. cypriaca, O. pleschanka and
O. h. melanoleuca. With a relative high posterior probability value (0.98), O. cypriaca clusters closer to O. h.
melanoleuca from Iran and Israel (on migration) and
O. pleschanka from Iran than to O. pleschanka obtained
from Kazakhstan, Russia, Mongolia and wintering areas in
East Africa (Kenya). The scenario suggests that O. cypriaca
might be either a relatively young taxon, which is yet
behavioural distinct, but genetically still similar to its sister
populations on the mainland. Alternatively, we may
assume a close relationship as an indication for potential
ongoing hybridisation processes involving all three forms.
Keywords Cytochrome oxidase subunit 1 gene Dummy presentation Oenanthe cypriaca Playback Pre-zygotic isolation Species recognition
Zusammenfassung
Communicated by Jon Fjeldså.
C. Randler (&)
University of Education Heidelberg,
Im Neuenheimer Feld 561-2, 69120 Heidelberg, Germany
e-mail: [email protected]
M. I. Förschler F. Bairlein
Vogelwarte Helgoland, Institut für Vogelforschung,
An der Vogelwarte 21, 26386 Wilhelmshaven, Germany
J. Gonzalez M. Wink
Department of Biology, Institute of Pharmacy and Molecular
Biotechnology, University of Heidelberg, Im Neuenheimer Feld
364, 69120 Heidelberg, Germany
M. Aliabadian
Department of Biology, Faculty of Science,
Ferdowsi University of Mashhad, Mashhad, Iran
Phylogeographie, Taxonomie und präzygote Isolation
beim Zypernsteinschmätzer Oenanthe cypriaca
Endemismus auf Inseln ist ein bekanntes Phänomen. Der
Zypernsteinschmätzer Oenanthe cypriaca brütet nur auf
Zypern und wurde lange Zeit als Unterart des Nonnensteinschmätzers O. pleschanka bezeichnet, obwohl einige
Autoren besonders aufgrund des Gesangs ihn als eigene Art
betrachteten. In dieser Studie untersuchten wir die mitochondrielle Nukleotidensequenz der Cytochrome oxidase
subunit 1 um das phylogeographische Muster und die
genetische Struktur von O. cypriaca zu bestimmen. Zusätzlich
wurden noch Playbackexperimente und Modellpräsentationen durchgeführt (dummies). Dies sollte einen Hinweis auf
die präzygote Isolation geben. Die Studie fand im März/April
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2008 und Mai 2009 statt. Verschiedene Playbacks wurden
benutzt: konspezifischer Gesang und heterospezifischer von
Mittelmeersteinschmätzer O. h. hispanica/melanoleuca,
O. pleschanka und Felsensteinschmätzer O. finschii. Experimentelle Dummy Präsentationen beinhalteten Modelle von
O. cypriaca, O. pleschanka und sowohl eine dunkle als auch
eine helle Morphe von O. h. melanoleuca. O. cypriaca reagierte signifikant stärker auf konspezifischen Gesang und
konspezifische Modelle als auf O. pleschanka und die anderen
Oenanthe-Arten. 679 Basen Paare (bp) der mitochondriellen
Cytochrome c oxidase subunit 1 (CO1) wurden untersucht.
Die Maximum-Likelihood und Bayesian Inference Analysen
unterstützten die nahe Verwandtschaft zwischen Oenanthe
cypriaca, O. pleschanka und O. h. melanoleuca. Dieselbe
Analyse allerdings zeigte interessanterweise, dass—mit hoher
bootstrap Wahrscheinlichkeit—O. cypriaca ein Cluster bildet, indem die Art näher mit O. h. melanoleuca aus Iran und
Israel (vom Durchzug) und O. pleschanka aus Iran als mit
O. pleschanka aus Überwinterungsgebieten in West Afrika
(Kenia) verwandt ist. Die Daten dieser Studie unterstützen
teilweise den Status des Zypernsteinschmätzers als eigenständige Art, aber die molekulare Analyse zeigt, dass die
Artbildung wahrscheinlich noch sehr jung ist oder Hybridisationsprozesse stattfinden.
Introduction
Geographic isolation—especially on islands—is one main
factor of intraspecific differentiation, speciation and endemism
(Price 2008), and it is interesting to see the historical change in
how island forms are being treated by taxonomists. Under the
broad biological species concept, many island forms have been
treated as subspecies of widespread species of the adjacent
mainland, under the assumption that they would probably
hybridise if they were to meet. In more recent times, the trend is
rather to recognize clearly diagnosable island forms as separate
evolutionary units, and to give them status as distinct species.
On a European scale, island endemism and evolution of discrete
species is reported from Macaronesia (see e.g. Päckert and
Martens 2004; Dietzen et al. 2003, 2005, 2008a, b; Gonzalez
et al. 2009) and from the Mediterranean (e.g. Brambilla et al.
2008; Förschler et al. 2009).
On Cyprus, the Cyprus Wheatear Oenanthe cypriaca has
been considered as distinct species by different authors
because of morphometric measurements, less pronounced
sexual dichromatism and significantly different song than
the Pied Wheatear O. pleschanka (Homeyer 1884; Christensen 1974; Sluys and van den Berg 1982; Bergmann
1983; Flint and Stewart 1992; Flint 1995; Förschler et al.
2010), while other authors retained it as a subspecies of
O. pleschanka (e.g. Panov 2005). Behavioural observations
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J Ornithol (2012) 153:303–312
and ecological studies by Randler and Crabtree (2010)
provided additional evidence for the splitting, and in a recent
work it has been shown that both forms are very well separated by several morphological traits (Förschler et al. 2010).
However, molecular studies have not been carried out to
support the morphological, behavioural and bioacoustic
evidence. Above and beyond the molecular analysis, prezygotic isolation—a key factor of the biological species
concept—should also provide further evidence for species
isolation (e.g., in assortative mating; Randler 2008). As there
are no overlapping breeding areas with other Oenanthe
species, hybridisation—which is common in Oenanthe
species (Cramp 1988; Panov 2005; Randler 2006; Aliabadian et al. 2007)—could not be assessed as a pre-requisite for
the biological species concept (e.g. Randler 2002, 2004), and
we therefore used playback experiments and model
(dummy) presentation to simulate the intruding of heterospecifics. In contrast to the Macaronesian and many tropical
island species, the Cyprus Wheatear is a migratory species,
and thus meets congeners during migration and on the winter
quarters, and this makes a thorough study of potential gene
flow and reproductive isolation highly interesting.
In this study, using mitochondrial nucleotide sequences
of the cytochrome oxidase subunit 1 gene, we aim to
analyse the phylogeographical and genetic structure of the
Cyprus Wheatear compared to closely related taxa such as
the Pied Wheatear and the two recognised subspecies of
Black-eared Wheatear (O. h. hispanica and O. h. melanoleuca). By using playbacks and dummy presentations,
we further assess the pre-zygotic isolation in O. cypriaca.
The strength of the present study is the combination of
molecular data with aspects of pre-zygotic isolation.
Methods
Study area
Cyprus is an island in the south-eastern Mediterranean,
approximately 225 km long and 100 km wide. It is the
third largest island in the Mediterranean (9,250 km2) after
Sicily and Sardinia (Stagg and Hearl 1997), and lies less
than 100 km from Turkey, and less than 200 km from
Syria and Lebanon at 34°330 –35°420 N, 32°160 –34°360 E
(Jones 2006). The island is dominated by two mountain
ranges, the Troodos mountains in the south rising to
1,961 m and further north, the Kyrenia range rising to
1,024 m. Cyprus has an extreme Mediterranean climate
with long, very hot, dry summers and cool, wet, changeable
winters (Flint and Stewart 1992). The island has a variety
of natural vegetation; 18% of the island is woodland (Stagg
and Hearl 1997; Förschler and Randler 2009).
J Ornithol (2012) 153:303–312
DNA isolation and sequencing of the mitochondrial
CO1 gene
Samples from Oenanthe species were genetically analysed
from different populations located in Cyprus, Israel, Asia and
Africa (for details, see Table 1, and Fig. 2, below). The DNA
was obtained from blood or growing feathers. Total DNA was
isolated using standard proteinase K (Merck, Darmstadt) and
phenol/chloroform procedures (Sambrook et al. 1989).
We amplified a fragment of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene using the primers
passerF1 and passerR1 (Lohman et al. 2009). The PCR
amplifications were performed in 50-ll reaction volumes
containing 19 PCR buffer (Bioron, Ludwigshafen),
100 lM dNTPs, 0.2 units of Taq DNA polymerase (Bioron), 200 ng of DNA and 5 pmol of primers. Optimal
annealing temperature was found by gradient PCR in a
Tgradient thermocycler (Biometra). Thermal cycling was
performed under the following conditions: 5 min at 94°C,
followed by 35 cycles of 40 s at 94°C, 40 s at 52.0°C,
1 min at 72°C and a final extension at 72°C for 10 min.
PCR products were precipitated with 4 M NH4Ac and
ethanol (1:1:6) and centrifuged for 15 min (13,000 rpm).
Sequencing was performed using a ABI 3730 automated
capillary sequencer (Applied Biosystems) with the ABI
Prism Big Dye Terminator Cycle Sequencing Ready
Reaction Kit version 3.1 by STARSEQ (Mainz, Germany).
In order to confirm observed mutations, both strands of
each sample were sequenced. All sequences generated in
this study have been deposited in GenBank under accession
numbers HM126495–HM126527.
The nucleotide sequences were aligned manually with
BIOEDIT version 7.0.9.0 (Hall 2004). No internal stop codons
or frame-shifts were observed in the sequences that were
translated entirely by using the chicken mitochondrial code.
The typical mitochondrial pattern of predominantly third
codon position substitutions was evident (Moore and DeFilippis 1997). Basic statistics and average uncorrected p distances were calculated with MEGA version 4.0 (Tamura
et al. 2007). Phylogenetic trees were reconstructed using
maximum likelihood (ML) in PAUP* version 4.0b10a
(Swofford 2002) and Bayesian inference (BI) in MRBAYES
version 3.1.2. (Ronquist and Huelsenbeck 2003). Following
Outlaw et al. (2010), the trees were rooted using several
species of stonechats (Saxicola), rock-thrushes (Monticola),
the Orange-flanked Bush-robin (Tarsiger cyanurus), two
species of nightingales (Luscinia luscinia and L. megarhynchos) and the Rufous-backed Redstart (Phoenicurus
erythronotus) as outgroups (see Table 1).
We explored the model of sequence evolution that fits
the data best with MODELTEST version 3.7 (Posada and
Crandall 1998) and MRMODELTEST version 2.3
(Nylander 2004). ML heuristic searches were performed
305
with closest stepwise sequence additions, tree-bisectionreconnection branch-swapping (TBR), ‘multrees’ option
and the best model found with MODELTEST. In the ML
analyses, the robustness of each node was assessed by
1,000 bootstrap replicates. For BI analyses, two independent runs of 8,000,000 generations each were performed
along with four Markov chains. Trees were sampled every
500 generations and the first 4,000 samples were discarded
as ‘burn-in’. The evolutionary models selected for BI and
ML analyses were GTR ? G ? I and HKY ? I ? G,
respectively.
Dummy presentation
Dummies were presented in eight different territories of
O. cypriaca. The dummies were identical in size and structure, but differed in their coloration (producer: artfauna). The
four male dummies represented three species (Fig. 1),
O. cypriaca, O. pleschanka, and O. h. melanoleuca. The
latter species was presented in two distinct morphs, the lightthroated and the dark throated morph. O. pleschanka is
closest in coloration to O. cypriaca, and the light-throated
morph of O. h. melanoleuca differs most. Dummy presentations were carried out between 11 and 18 May 2009, when
weather conditions were good (no rain, less wind), between
0700 and 1300 hours local time. The dummy was placed
always at the same post (e.g. a large stone) where it was
widely visible to the territory owner. In each of the eight
territories, all four models were presented. To avoid any
carry-over or habituation effects, only one dummy per day
was presented in the respective territory. As the order of
presentations might also have an influence on the response,
the order of dummy presentation was balanced between
territories, i.e. every dummy type (species) was presented in
two territories as first model, in two as second, in two as third
and in two as fourth model. For each male territory owner,
the following variables were collected: response latency,
minimum distance to the dummy, number of flights over the
dummy, time spent near dummy (\10 m).
Playback presentation
Playbacks were carried out in April 2008 and May 2009,
using the conspecific song as well as heterospecific songs
of O. h. hispanica/melanoleuca, O. pleschanka, and
O. finschii. The latter served as some kind of control
because the species is not closely related to the three other
Oenanthe (Aliabadian et al. 2007), but a regular winter
visitor to Cyprus. All stimuli were standardised on 30 s in
2008 and to 60 s in 2009. The calls for 2008 were obtained
from different commercial sound recordings (Roche 1995;
Kosmos-Verlag 2002; Schulze 2003), and for 2009 created
from own recordings of O. cypriaca from 2008 (by C.R.),
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J Ornithol (2012) 153:303–312
Table 1 Taxa sequenced in this study
Taxon
Table 1 continued
Collection
codeb
GenBank
accession
number
Origin
Taxon
Oenanthe cypriacaa
51654
HM126495
Cyprus
a
Oenanthe cypriaca
51655
HM126496
Cyprus
Oenanthe cypriacaa
51656
HM126497
Oenanthe cypriacaa
51658
a
Oenanthe cypriaca
Oenanthe cypriacaa
Collection
codeb
GenBank
accession
number
Origin
Oenanthe leucopyga
DQ683508
Morocco
Oenanthe pleschanka
DQ683507
Iran
Cyprus
Oenanthe pleschanka
DQ683506
Iran
HM126498
Cyprus
O. oenanthe seebohm
DQ683505
Morocco
51660
HM126499
Cyprus
O. oenanthe libanotica
DQ683504
Iran
51662
HM126500
Cyprus
O. oenanthe libanotica
DQ683501
Iran
Oenanthe cypriacaa
51664
HM126501
Cyprus
O. moesta moesta
DQ683500
Morocco
a
Oenanthe cypriaca
51666
HM126502
Cyprus
O. lugens persica
DQ683497
Iran
Oenanthe cypriacaa
51670
HM126503
Cyprus
Oenanthe isabellina
DQ683495
Iran
Oenanthe hispanica
53444
HM126504
Niger
O. hispanica melanoleuca
DQ683489
Iran
Oenanthe hispanica
53445
HM126505
Mali
O. finschii barnesi
DQ683487
Iran
Oenanthe hispanica
53446
HM126506
Mali
O. deserti homochroa
DQ683485
Morocco
Oenanthe hispanica
53448
HM126507
Mali
O. deserti deserti
DQ683484
Iran
Oenanthe hispanica
53449
HM126508
Mauritania
Oenanthe chrysopygia
DQ683481
Iran
Oenanthe hispanica
53451
HM126509
Mauritania
Oenanthe alboniger
DQ683480
Iran
Oenanthe hispanica
53452
HM126510
Mauritania
Saxicola insignis
GQ482619
Mongolia
Oenanthe hispanica
53453
HM126511
Mauritania
Saxicola maura
GQ482621
Zaliv, Russia
Oenanthe hispanica
53454
HM126512
Mauritania
Saxicola rubetra
GQ482628
Russia
Oenanthe hispanica
53455
HM126513
Mauritania
Saxicola rubicola
GQ482633
Oenanthe hispanica
53456
HM126514
Mauritania
Akhmetovskaya,
Russia
Oenanthe hispanica
53457
HM126515
Mauritania
S. torquata axillaris
FJ657469
Kenya
GQ482168
Mongolia
Oenanthe hispanica
53467
HM126516
Morocco
Monticola gularis
O. hispanica melanoleuca
53468
HM126517
Israel
Monticola saxatilis
GQ482172
Kazakhstan
Israel
Tarsiger cyanurus
GQ482758
Mongolia
DQ683476
Sweden
O. hispanica melanoleuca
53469
HM126518
Oenanthe pleschanka
53458
HM126519
Kenya
Luscinia luscinia
Oenanthe pleschanka
53459
HM126520
Kenya
Luscinia megarhynchos
DQ683477
Iran
Kenya
Phoenicurus erythronotus
GQ482382
Russia
a
Oenanthe pleschanka
53460
HM126521
Oenanthe pleschanka
53461
HM126522
Kenya
Oenanthe pleschanka
53462
HM126523
Kenya
Oenanthe pleschanka
53463
HM126524
Kenya
Oenanthe pleschanka
53464
HM126525
Kenya
Oenanthe pleschanka
53465
HM126526
Kenya
Oenanthe pleschanka
53466
HM126527
Kenya
Oenanthe deserti
GQ482258
Mugur Aksy,
Russia
Oenanthe pleschanka
GQ482271
Kazakhstan
Oenanthe pleschanka
GQ482272
Mongolia
Oenanthe pleschanka
GQ482273
Mongolia
Oenanthe pleschanka
GQ482274
Novorossiysk,
Russia
Oenanthe pleschanka
GQ482275
Mugur Aksy,
Russia
Oenanthe oenanthe
AY666389
Canada
Oenanthe oenanthe
DQ433051
Iceland
O. picata picata
DQ683509
Iran
123
Sampled in breeding range. The O. h. hispanica and O. h. melanoleuca
were sampled during migration and in their winter quarters
b
The collection code corresponds to those of the Institute of Pharmacy
and Molecular Biotechnology (IPMB), University of Heidelberg
and from recordings of O. h. melanoleuca from the British
Sound Library. For every species, different stimulus tapes
were created to avoid pseudo-replication (Kroodsma 1989).
The calls were digitally edited to minimise disturbing noises
and to erase songs or calls of other bird species using Avisoft
SASLab Pro 4.3. Afterwards, the calls were copied to an
analogous tape using a Grundig 437 CD player and AIWA
CX-Z87M cassette recorder to produce the playback tapes.
Calls were broadcast using a small portable Toshiba MCR 103
cassette recorder. The different playback tapes were standardised at ca. 65–75 dB measured at 1 m distance from the
speaker using a digital sound-level meter with A-level
weighting (PeakTech 5035). In 2008, there were n = 8 dif-
J Ornithol (2012) 153:303–312
307
Fig. 1 Dummy models of the four types used for the experiments. From left to right O. cypriaca, O. pleschanka, O. h. melanoleuca (dark), and
O. h. melanoleuca (light)
ferent playback stimuli available for O. finschii, n = 9 for O.
pleschanka, n = 17 for O. h. hispanica/melanoleuca, and
n = 8 for O. cypriaca. The O. hispanica playbacks were from
both subspecies, O. h. hispanica and O. h. melanoleuca, and
from populations that had not been assigned subspecific status. In 2009, there were n = 15 playback tapes for O. cypriaca, and n = 13 for the eastern subspecies O. h. melanoleuca.
During every trial, the focal individual received two playbacks—one conspecific and one heterospecific (matched pair
samples). However, to avoid carry-over effects in playback
designs (den Hartog et al. 2007), in half the trials the conspecific stimulus was played first followed by the heterospecific song, and in the other half it was vice versa. Responses to
the playback tapes were coded in the following manner:
0 = no visible or acoustic response, 1 = weak response
(changing/altering of song, calling, or dipping, but no
approach), 2 = medium response (approaching the speaker
but less than 10 m, or 1–2 flights in the direction of the
speaker), 3 = strong response approaching the speaker up to
10 m at least once), 4 = very strong response (two approaches nearer than 10 m or minimum distance below 5 m).
Statistical analysis
For the playback and dummy presentation, non-parametric
tests were used: Wilcoxon-test for the comparison of two
dependent observations, and Mann–Whitney U test for
two independent observations. To compare more than two
different treatments, Friedman test for dependent and
Kruskal–Wallis for independent observations were used.
Further, the responses to the dummy presentation were
subjected to a factor analysis to create a single response
variable. Three variables (minimum distance, overflights,
time spent less than 10 m near the dummy) were log10transformed after the addition of one. Afterwards, these
variables and response latency were z-transformed and
subjected to a principal component analysis with varimax
rotation.
Results
Phylogenetic analysis
We obtained 679 base pairs (bp) of the mitochondrial
cytochrome c oxidase subunit 1 (CO1) gene. ML and BI
analyses support the close relationship between O. cypriaca, O. pleschanka and O. h. melanoleuca (Fig. 2). With a
relative high posterior probability value (0.98), O. cypriaca
clusters closer to migrating O. h. melanoleuca from Iran
and Israel and O. pleschanka from Iran than to O. pleschanka obtained from Kazakhstan, Russia and Mongolia and
wintering areas in East Africa (Kenya). Uncorrected p
distance values between O. cypriaca and O. pleschanka
from Kenya consist of 0.5%. O. hispanica samples from
different West African wintering localities such as Mali,
Morocco, Niger and Mauritania cluster separately in different branches of the tree (Fig. 2). The birds, which winter
in these areas and belong phenotypically to the sub-species
O. h. hispanica, are probably of Central and Western
Mediterranean origin (e.g. Italy, France and Spain). The
phylogenetic relationships among other Oenanthe species
at the base of the tree have been previously recovered and
discussed in Aliabadian et al. (2007) and Outlaw et al.
(2010).
Dummy presentation
Friedman tests indicated significant differences in the
response of O. cypriaca males towards the four different
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J Ornithol (2012) 153:303–312
Fig. 2 Bayesian tree based on
679 bp of the co1 gene.
Posterior probability values
([0.90) are indicated for each
node. The origin of samples is
indicated in Table 1
O. hispanica melanoleuca DQ683489 Iran
O. pleschanka DQ683507 Iran
O. pleschanka DQ683506 Iran
O. cypriaca 51654 Cyprus
O. hispanica melanoleuca 53469 Israel
O. hispanica melanoleuca 53468 Israel
0.98 O. cypriaca 51670 Cyprus
O. cypriaca 51666 Cyprus
O. cypriaca 51664 Cyprus
O. cypriaca 51662 Cyprus
O. cypriaca 51660 Cyprus
O. cypriaca 51658 Cyprus
O. cypriaca 51656 Cyprus
O. cypriaca 51655 Cyprus
O. pleschanka GQ482271 Kazakhstan
O. pleschanka GQ482275 Mugur Aksy Russia
0.98
O. pleschanka GQ482274 Novorossiysk Russia
1.00
O. pleschanka GQ482273 Mongolia
O. pleschanka GQ482272 Mongolia
O. pleschanka 53461 Kenya
0.97
O. pleschanka 53466 Kenya
O. pleschanka 53463 Kenya
O. pleschanka 53458 Kenya
O. pleschanka 53465 Kenya
O. pleschanka 53464 Kenya
O. pleschanka 53462 Kenya
O. pleschanka 53460 Kenya
O. pleschanka 53459 Kenya
0.99 O. hispanica 53445 Mali
O. hispanica 53467 Morocco
1.00
O. hispanica 53448 Mali
1.00
O. hispanica 53444 Niger
O. hispanica 53451 Mauritania
0.93
O. hispanica 53455 Mauritania
O. hispanica 53454 Mauritania
O. hispanica 53446 Mali
O. hispanica 53457 Mauritania
O. hispanica 53456 Mauritania
O. hispanica 53453 Mauritania
O. hispanica 53452 Mauritania
O. hispanica 53449 Mauritania
1.00
O. chrysopygia DQ683481 Iran
1.00
1.00
O. lugens persica DQ683497 Iran
0.99
O. finschii barnesi DQ683487 Iran
O. alboniger DQ683480 Iran
O. leucopyga DQ683508 Morocco
1.00
O. picata picata DQ683509 Iran
O. moesta moesta DQ683500 Morocco
0.95 O. oenanthe AY666389 Canada
O. oenanthe DQ433051 Iceland
1.00
O. oenanthe libanotica DQ683504 Iran
1.00
O. oenanthe libanotica DQ683501 Iran
0.94 O. oenanthe seebohmi DQ683505 Morocco
O. isabellina DQ683495 Iran
O. deserti deserti DQ683484 Iran
1.00
O. deserti homochroa DQ683485 Morocco
O. deserti GQ482258 Mugur Aksy Russia
1.00
Saxicola rubicola GQ482633 Akhmetovskaya Russia
1.00
Saxicola torquata axillaris FJ657469 Kenya
0.95
Saxicola maura GQ482621 Zaliv Russia
1.00
Saxicola insignis GQ482619 Mongolia
Saxicola rubetra GQ482628 Russia
1.00
Monticola gularis GQ482168 Mongolia
Monticola saxatilis GQ482172 Kazakhstan
Tarsiger cyanurus GQ482758 Mongolia
1.00
Luscinia luscinia DQ683476 Sweden
Luscinia megarhynchos DQ683477 Iran
Phoenicurus erythronotus GQ482382 Russia
1.00
0.1
0.98
0.91
models in minimum distance to the dummy (v2 = 8.52,
df = 3, P = 0.036), time spent \ 10 m from the dummy
(v2 = 12.67, df = 3, P = 0.005), and number of overflights (v2 = 16.63, df = 3, P = 0.001). Response latency
did not differ significantly (v2 = 1.75, df = 3, P = 0.625).
The PCA produced one factor with an Eigen-value greater
123
than one (Eigen-value: 2.93; 73.3% variance explained).
Factor loadings were -0.96 concerning minimum distance,
0.88 time spent near the dummy, 0.88 (overflights), and
-0.67 concerning response latency. O. cypriaca responded
stronger towards the conspecific model (Fig. 3) than to the
O. pleschanka model (T = 2.77, df = 7, P = 0.028), the
J Ornithol (2012) 153:303–312
2
P=0.028
309
P=0.032
P=0.006
Table 2 Responses of O. cypriaca males towards playback
presentations
n
1
Z
P
-2.591
0.010
2008
Stimulus
O. pleschanka
0
-1
Negative ranks
12
Positive ranks
2
Ties
3
Total
17
O. hispanica (both subspecies and undetermined populations)
-2
O. cypriaca
O. pleschanka
O. h. melanoleuca
(dark)
O. h. melanoleuca
(light)
Fig. 3 Responses of O. cypriaca towards four dummy presentations
(stimuli; based on a PCA, see ‘‘Methods’’). Significance is indicated
between the responses towards the conspecific and each of the three
heterospecific dummies
dark-throated O. h. melanoleuca model (T = 2.67, df = 7,
P = 0.032) and the light-throated O. h. melanoleuca model
(T = 3.92, df = 7, P = 0.006). However, responses did
not differ significantly between the three heterospecific
models (P [ 0.12; Fig. 3).
Playbacks
In the matched pair comparisons, O. cypriaca responded
stronger to conspecific playbacks (Table 2) than to heterospecific playbacks of O. h. hispanica/melanoleuca,
O. pleschanka and O. finschii. Responses to playbacks
were significantly different between the four stimuli
(Kruskal–Wallis-Test: v2 = 20.40, df = 3, P \ 0.001;
based on the first playback for every individual/trial;
Fig. 4). In May 2009, for the heterospecific playbacks, only
songs of the O. h. melanoleuca were used because the
response in 2008 was strongest towards O. h. hispanica/
melanoleuca. Similarly to 2008, O. cypriaca reacted most
strongly towards conspecific playbacks (Table 2), and in
only 2 out of 15 cases did a response towards O. h. melanoleuca playback occur. The response towards playbacks
was weaker in May 2009 than in April 2008.
Discussion
Negative ranks
11
Positive ranks
3
Ties
1
Total
15
0.025
-1.633
0.102
-2.050
0.040
O. finschii
Negative ranks
3
Positive ranks
0
Ties
0
Total
3
2009
O. h. melanoleuca
Negative ranks
6
Positive ranks
1
Ties
Total
8
15
Statistics based on Wilcoxon test for matched pairs (details see
‘‘Methods’’)
4
3
2
1
0
O. cypriaca
The results of the behavioural experiments support the prezygotic isolation of O. cypriaca towards O. pleschanka and
O. h. melanoleuca because O. cypriaca responded significantly more strongly towards conspecific playback and
dummy presentations than to heterospecific stimuli.
-2.249
O. pleschanka
O. hispanica
O. finschii
Fig. 4 Responses of O. cypriaca towards conspecific and different
heterospecific playbacks. 0 no response, 1 weak response, 2 medium
response, 3 strong response, 4 very strong response. Data from 2008,
O. hispanica playbacks from different and unknown subspecies (see
‘‘Methods’’)
123
310
Responses to playbacks are generally seen as a useful tool
in avian systematics but must be treated with some caution
when considering allopatric taxa. It is to be expected that a
male reacts more strongly to a visual or vocal stimulus that
it has been imprinted on than to an alien stimulus (e.g. an
alloptric taxon). However, although the Oenanthe
wheatears are allopatric with regard to their breeding area,
O. h. melanoleuca is a regular spring migrant on Cyprus
(Randler and Crabtree 2010). Therefore, the species are
familiar with each other and cannot be considered as
clearly allopatric. Further, as O. cypriaca is migratory there
might be some overlapping in migratory (Israel) and wintering areas (Kenya), thus the heterospecific recognition
should be at least partially developed. This makes the situation of O. cypriaca different from many other island
endemics, especially from tropical archipelagos and from
Macaronesia because those are sedentary species. O. cypriaca might be allopatric from the breeding range but is
sympatric with related taxa considering winter quarters.
However, detailed studies of the coexistence of the
Oenanthe species on migration and in the winter quarters
are scarce (Leisler et al. 1983; Randler and Crabtree 2010).
Nevertheless, it is remarkable that this island population
seems to maintain its integrity despite the opportunity to
mix with mainland populations during their life cycle
which strongly supports a pre-zygotic barrier.
From the molecular viewpoint, we are unable to clearly
separate O. cypriaca from O. pleschanka and O. h. melanoleuca. The small genetic distance is in agreement with
intra-population divergence in many other passerines
(Alström et al. 2007, 2008), and this contradicts rather than
supports the species status of O. cypriaca. Another interesting aspect is the non-monophyly which is strongly
supported by mitochondrial data (Olsson et al. 2005). The
molecular tree surprisingly indicates that migrating O. h.
melanoleuca from Israel and O. pleschanka from Iran
clustered together with O. cypriaca, which might be an
indicator for the retention of ancestral polymorphism or
introgression. There seems to be gene flow between O. cypriaca and O. h. melanoleuca, and between O. h. melanoleuca
and O. pleschanka (Aliabadian et al. 2007). The latter, however, is well-known (Panov 2005). Gene flow between
O. cypriaca and O. h. melanoleuca might be a result of
introgression, although there is no firm evidence of this.
Introgression may have occurred at different temporal stages,
and it could be either recent or have taken place a long time
ago (see below). An alternative hypothesis for the pattern
might therefore be retention of ancestral polymorphisms.
Hybridisation is likely among Oenanthe species, especially between O. pleschanka and O. h. melanoleuca
(Panov 2005; Aliabadian et al. 2007; Randler 2008), which
show a wide hybrid zone in Iran. Although there are no
breeding records from O. cypriaca outside Cyprus, it may
123
J Ornithol (2012) 153:303–312
be possible that the species also breeds on the southern
coast of Turkey, because the species apparently has some
vagrancy potential (Förschler et al. 2010) and, recently, we
have suggested probable breeding of O. h. melanoleuca on
Cyprus (Randler and Crabtree 2010), which may also
facilitate hybridisation between the taxa.
We used different measurements to assess the taxonomic status of O. cypriaca (see Alström et al. 2007,
2008), and we have to conclude that the mitochondrial data
do not support species status of O. cypriaca, and even not
of O. h. melanoleuca and O. pleschanka, while, surprisingly, the data support different forms within the western
O. h. hispanica clade. Alström et al. (2008) discuss the
limits of assigning species rank in allopatric taxa. Under a
‘morphological’ species definition, O. cypriaca might be a
distinct species (Christensen 1974; Aliabadian et al. 2007;
Förschler et al 2010), while under a ‘phylogenetic’ species
definition it might not. Under a ‘biological’ species definition, the taxonomic status of O. cypriaca seems clearer
because the song of O. cypriaca is different compared to
the other three forms (Sluys and van den Berg 1982;
Bergmann 1983). Despite the mitochondrial evidence
against species status, we propose species status for
O. cypriaca because of the behaviour towards dummies
and playbacks, the strikingly different song (Bergmann
1983), and different morphometrics (Kaboli et al. 2006).
O. cypriaca differs from O. pleschanka in 14 characters of
external morphology (Förschler et al. 2010). Given this
evidence, we suppose that O. cypriaca will maintain its
genetic and phenotypic integrity in the future, which should
be a significant criterion for assigning species rank (Helbig
et al. 2002). An alternative would be to treat O. pleschanka,
O. h. melanoleuca and O. cypriaca as populations of one
single species. The results emphasise the importance of
dense taxon sampling in intrageneric phylogenetic studies
as requested by Olsson et al. (2005), and, we therefore
suggest further sampling of the hispanica–melanouleuca–
cypriaca–pleschanka complex in all breeding areas.
Acknowledgments The study was supported by the Forschungskommission of the Deutsche Ornithologen-Gesellschaft (DO-G). The
research presented here was carried out under permit licence 08/2007
and 4/2009 of the Game Fund of the Ministry of Interior Cyprus and
complies with German and Cyprus law. We are grateful to Alan
Crabtree, Ivan Maggini, Benjamin Metzger and Reuven Yosef for
help during field work. Per Alström and an anonymous reviewer
provided helpful comments on the manuscript.
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